Worldwide 33.4 million people lived with HIV/AIDS in 2008, with 2.7 million new infections, and 2 million related deaths. In the US, 1.4 million people had HIV/AIDS in 2008, with 55,000 new infections, and 25,000 related deaths. Certain disadvantaged or vulnerable populations bear a disparate share of this burden, including women and children globally, and Black American (BA) and Hispanic minorities in USA. BAs acquired 46% of new infections in 2006, despite comprising only 13% of the population. Among females, BAs share an even more disproportionate burden, acquiring 61% of all new female HIV infections in 2006. For 2006 male infections, 76% were MSM. In these vulnerable groups, drug use including amphetamines, opiates, alcohol, and cocaine, is associated with high-risk sexual behavior, and accelerates progression of and mortality from HIV. Drug use is also an independent risk factor for seroconversion, and predictive of disease progression, development of AIDS, and AIDS mortality. Thus it is increasingly important to understand impacts of comorbid drug abuse on HIV infection, particularly as it affects vulnerable populations and therapy development [1-10]. Some evidence implicates glycogen synthase kinase 3 (GSK3) as a possible regulator of peripheral HIV infection, and GSK3 also may mediate effects of some abused drugs, such as cocaine and methamphetamine. However there has been little investigation of exactly how or whether GSK3 directly affects HIV infection with and without comorbid drug abuse, particularly as regards key HIV tropic peripheral CD4+ T cells and macrophages. Hence, this proposal will investigate the unifying hypothesis that GSK3 activation increases endocytosis to enhance coreceptor mediated HIV infection of peripherally derived human CD4+ T cells and macrophages, and that this is the mechanism by which cocaine and amphetamines enhance HIV infection of these cell types. First we will determine if GSK3 transgene overexpression or knockdown directly affects HIV infection of CD4+ T cells and macrophages. Next, using fluorescent, immunocytochemical, and western blot assays for endocytosis, and siRNA knockdown of Rab5, we will determine whether GSK3 increases in HIV infection in CD4+ T cells and macrophages by enhancing Rab5-mediated endocytosis. In Aim 2, using similar approaches, we will first determine whether Meth or cocaine treatment increases GSK3 expression and activity, and endocytosis, in these cell types. We will further determine whether Meth or cocaine increase HIV infection of these cells, and whether these effects can be abrogated by manipulating GSK3 and/or Rab5 expression. In all of these studies, with and without cocaine and Meth, we will determine whether GSK3's effects on HIV infection of CD4+ T cells and macrophages can be ameliorated by the GSK3-inhibiting compounds lithium and AR-A014418, to determine whether GSK3 inhibition is a potentially viable therapeutic strategy for reducing viral load in HIV disease. Thus these studies will provide new insights into the molecular mechanisms regulating HIV infection of highly HIV-tropic peripheral immune cells, to identify new therapies for treating HIV disease with comorbid drug abuse. PUBLIC HEALTH RELEVANCE: This project explores new mechanistic theories that may help explain how HIV enters human immune cells to cause HIV disease. Better understanding of these disease mechanisms concerning how HIV causes infection of principal cell types involved in HIV, will help identify additional therapeutic targets for preventative intervention to treat HIV and AIDS.